Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.


March 2021
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. ]. Details of the relative expression are also provided in Figure 1 and Supplementary Dataset 1. The updated Chlamydomonas reinhardtii genome and gene annotation for the RNA sequencing and DNA insertion analysis were downloaded from JGI Phytozome v13, Chlamydomonas reinhardtii v5.6 [https://data.jgi.doe.gov/refine-download/phytozome?organism=Creinhardtii&expanded=281]. All sequence reads for the insertion site were deposited in the NCBI SRA with BioProject accession code PRJNA767110 [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA767110]. All the relevant data are available from the corresponding author upon reasonable request. The related statement is included in the manuscript.
No sample size was predetermined. For the RNA sequencing, a pool of total RNA from six biological replicates (cells in each culture were totally harvested) was sequenced for each three condition (i.e., ambient (atmospheric), pH 5.0 acidic, and 20% high CO2 conditions), respectively, to reduce the amount of biological variability. For experiments involving RT-PCR, growth assessment, photosynthetic rate assay, ELISA, intracellular pH measurement, in vitro ATPase activity assay, etc., n=2 was chosen as the minimal replicate number. For the flow cytometry analysis, the fluorescence measurement lasted until 10,000 events occurred. Sample size was determined to be adequate based on the magnitude and consistency of measurable differences among groups.
No data were excluded from the analyses.
All experiments were performed with at least two biological replicates, including RT-PCR, growth assessment, photosynthetic rate assay, ELISA, etc. Two quantitative biochemical experiments, including intracellular pH measurement and in vitro ATPase activity assay (ATPasemediated ATP hydrolysis performance test), were conducted in quadruplicate to secure the reliability (indicated in the manuscript and the corresponding legends). All replication attempts were successful.
No specific method of randomization was used for the generation of samples or cell experiments since all algal cells in each genotype are identical. For any experiments in which aliquots are needed, they were sampled randomly from each cell line culture. Confocal imaging were selected randomly from three biologically independent samples.
Blinding was not used in this study. The investigators were not blinded during data collection and analyses.
In this study, anti-plasma membrane H+-ATPase (Supplier: Agrisera, Sweden; Catalog number: AS07 260; Clonality: Polyclonal; Host: Rabbit) was used as the primary antibody for detecting the heterologously expressed PMA protein and natively expressed PMA with Western blotting at the same time. Anti-beta subunit of ATP synthase (Supplier: Agrisera, Sweden; Catalog number: AS05 085; Clonality: Polyclonal; Host: Rabbit) was used as the primary antibody for detecting the endogenous loading control (i.e., beta subunit of ATP synthase). For both cases, HRP conjugated goat anti-rabbit IgG (H+L) secondary antibody (Supplier: Agrisera, Sweden; Catalog number: AS09 602) was used as the secondary antibody.